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Kinetics of oxidation and reduction of high-potential iron-sulfur proteins with nonphysiological reactants
Abstract:
We have investigated the kinetic and equilibrium oxidation-reduction properties of HIPIP's from four sources: Chromatium vinosum, Thiocapsa pfennigii, Rhodopseudomonas gelatinosa, and a paracoccus sp. These studies focused on the interaction of the various HIPIP's and the iron hexacyanides as a function of ionic strength, pH, and temperature. On the basis of the kinetic and equilibrium data obtained and the known structrual properties of the various HIPIP's a number of conclusions concerning the mechanism of electron transfer by HIPIP can be inferred. (1) The oxidation and reduction of HIPIP by the iron hexacyanides show no kinetic complexity due to heterogeneity or changes in rate-limiting step. (2) The kinetic and equilibrium studies (oxidation-reduction potential) are in good agreement, although a rapid binding equilibrium between HIPIP and the iron hexacyanides is possible. (3) The kinetics of oxidation and reduction of the various HIPIP's studied do not correlate with their oxidation-reduction potentials. (4) The electrostatic interactions between a particular HIPIP and the iron hexacyanides appear to be influenced by the charge on the iron-sulfur cluster with specific amino acid side chains playing a significant but limited role in the interactions leading to electron transfer. (5) The iron-sulfur cluster charge is apparently distributed on the surface of the HIPIP molecule through a network of hydrogen bonds. (6) On the basis of an analysis of the known primary and tertiary structure of the HIPIP's studied, it is concluded that peptide backbone carbonyls and amino acid side chains for the amino acid sequence positions 46-49, 63-65, 78-82, 31-34, and 16-17 define the interaction site of HIPIP and the iron hexacyanides irrespective of the HIPIP source. This is a structural region at which the iron-sulfur cluster is near to the protein surface and in this respect resembles the active site in mitochondrial cytochrome c.